The present invention relates to modified microorganisms suitable for use as live vaccines. The present invention also relates generally to the use of modified microorganisms as biological vectors. The present invention further relates to vaccine compositions. In particular, the present invention relates to compositions suitable for inducing an immune response against RTX toxins.
Actinobacillus pleuropneumoniae (APP), is a member of the family Pasteurellaceae, and is the aetiological agent of porcine pleuropneumonia, an acute or chronic infection of pigs characterised by haemorrhagic, fibrinous and necrotic lung lesions (Pohl et. al., 1983; Kilian and Biberstein, 1984). The disease is highly contagious, and associated with all ages of growing pigs, resulting in severe economic losses to the swine industry. The direct mode of transmission of APP means that infection is more prevalent under intensive breeding conditions, fortunately the host range of APP is restricted to pigs reducing the potential sources of infection. To date, twelve serovars of APP have been identified worldwide (Serovars 1-12) with Serovars 1, 7 and 12 making up approximately 90% of Australian isolates (Kamp and Shope, 1964). A number of potential virulence factors have been identified including outer membrane proteins (Mulks and Thacker, 1988; Rapp and Ross, 1988), lipopolysaccharide (Udeze et al., 1987; Fenwick and Osburn, 1986), capsule (Inzana et al., 1988; Rosendale and Macinnes, 1990; Lenser et. al., 1988) and secreted toxins (Rycroft et al., 1991; Bhatia et al., 1991; Fedorka-Crey et al., 1990). The secreted toxins, or APX toxins, are members of the RTX toxin family (Frey et al., 1993, 1994).
RTX toxins are produced by a number of gram negative bacteria including Actinobacillus spp, Proteus vulgaris, Morganella morganii, Bordetella pertussis, Pasteurella haemolytica, and the most characterised of the group produced by E. coli (Welch, 1991) . All RTX toxins function by producing pores in the target cells, thereby interrupting osmotic balance, leading to rupture of the target cell. Although the mode of action is identical for RTX toxins their target cells vary greatly in type and cross species specificity. Structurally, this family of toxins are characterised by the presence of glycine rich repeat structures within the toxin that bind calcium and may have a role in target cell recognition and binding, a region of hydrophobic domains that are involved in pore formation, the requirement for post translational activation, and dependence on a C-terminal signal sequence for secretion (Reviewed: Coote, 1992).
At least three different APX toxins are produced by APP, designated APX1, APX2, and APX3. APX1 shows strong, and APX2 relatively low, haemolytic activity, both are cytotoxic and active against a broad range of cells of differing types and species (Frey and Nicolet, 1988; Rosendale et al., 1988; Kamp et al, 1991). APX3 is nonhaemolytic, but is strongly cytotoxic, with a host range including porcine alveolar macrophages and neutrophils (Rycroft et al., 1991; Kamp et al., 1991). No serovar of APP produces all three APX toxins, the majority produce two (APX1 and 2: Serovars 1, 5, 9, and 1 1; APX2 and 3: 2, 3, 4, 6, and 11) with a small number (APX1: 10, APX2: 7 and 12) producing only one APX (Frey and Nicolet, 1990; Frey et al., 1992,1993,1994; Kamp et al., 1991; Rycroft et al., 1991) The pattern of APX production appears to be associated with virulence, with those serovars producing APX1 and 2 being the most virulent (Frey et al., 1994; Komal and Mittal, 1990). Production and secretion of active RTX toxins requires the activity of at least four genes, C, A, B, and D. The A gene encodes the structural toxin, the C gene encodes the post-translational activator and the B and D genes encode proteins that are required for secretion of the activated toxin (Issartel et al., 1991; Welch, 1991; Felmlee et al., 1985). APX1 and 3 are encoded by operons that consist of the four contiguous genes (CABD), whilst the APX2 operon contains only the C and A genes, and in some cases remnants of the B gene. Secretion of APX2 is dependent on the activity of the APX1B and D gene products (Reviewed Frey et al., 1994).
Virulence analysis of spontaneous, and chemically induced, non-haemolytic mutants indicated a role of the APX toxins in virulence, which has recently been confirmed using transposon mutagenesis (Anderson et al., 1991; Gerlach et al., 1992; Inzana et al., 1991; Rycroft et al., 1991a,b; Tacon et al., 1993, 1994). Complete protection from disease and/ or carrier status cannot be obtained using vaccines comprising chemically inactivated bacteria, or purified subunit vaccines comprising outer membrane proteins, lipopolysaccharides, or capsule. In comparison complete protection from disease in mice was obtained following vaccination with purified APX combined with formalised whole cells, indicating a role for the APX toxins in protective immunity (Bhatia etal., 1991).
Vaccination against pleuropneumonia, resulting from APP infection of pigs, has utilised, to date, bacterins or subunit vaccines based on various components of the bacteria. Results obtained with inactive vaccines have offered, at best, homologous protection against the serovar used to prepare the vaccine material. Currently twelve known serovars of APP exist, of varying virulence, each requiring a different vaccine preparation. To date commercial vaccines have been formulated to contain a number of serovars, offering protection against the most frequently observed serovars in that geographic location. In contrast to inactive vaccines, natural infection with any one serovar offers protection against reinfection with any other serovar, indicating the potential of a live vaccine to offer cross protection against APP serovars.
It is an object of the present invention to alleviate one or more of the problems of the prior art.
Accordingly, in one aspect the present invention provides a modified microorganism which produces an RTX toxin, wherein said RTX toxin is partially or fully inactivated.
The term xe2x80x9cmodifiedxe2x80x9d includes modification by recombinant DNA techniques or other techniques such as chemical- or radiation- induced mutagenesis. Where recombinant DNA techniques involve the introduction of foreign DNA into host cells, the DNA may be introduced by any suitable method. Suitable methods include transformation of competent cells, transduction, conjugation and electroporation.
In a further embodiment of the present invention, there is provided a modified microorganism wherein an RTX toxin gene including an RTX structural gene and/or a post translational activator of the organism is partially or fully inactivated.
The term xe2x80x9cRTX toxin genexe2x80x9d as used herein the claims and description is intended to include those genes involved in the expression of an RTX toxin being a product of the RTX toxin gene. The genes included in the RTX toxin gene include the post translational activator gene (C), the structural gene (a), and the B and D genes which encode proteins that are required for secretion of the activated RTX toxin.
The term xe2x80x9cpartially or fully inactivatedxe2x80x9d as used herein the claims and description includes modification of a gene by recombinant DNA techniques including introduction and deletion of DNA from the gene including single or multiple nucleotide substitution, addition and/or deletion including full or partial deletion of the gene, using a target construct or plasmid segregation; and chemical induced-, radiation induced- or site specific mutagenesis.
The present applicants have found that a precursor of an RTX toxin has reduced toxic activity. Surprisingly, the present applicants have also found that the RTX toxin precursor is capable of inducing an immune response in an animal that offers cross protection against heterologous challenge with a microorganism which produces the RTX toxin.
Accordingly, in a preferred embodiment of the invention the inactivated RTX toxin is a precursor of an RTX toxin. The precursor may be an unprocessed expression product of an RTX structural gene. The RTX structural gene may be an RTX A gene.
The microorganism may be one which does not naturally produce an RTX toxin. The microorganism may be a bacterium, virus or fungus into which an RTX structural gene, such as an RTX A gene, has been introduced.
In a preferred embodiment, however, the microorganism is one which naturally produces an RTX toxin. I he microorganism which naturally produces an RTX toxin may be selected from Actinobacillus spp, Proteus vulgaris, Morganella morganii, Bordetella pertussis, Escherichia coli and Pasteurella haemolytica. In a preferred embodiment the microorganism is Actinobacillus spp. The Actinobacillus species may be Actinobacillus pleuropneumoniae (APP) and the RTX toxin may be an APX toxin. The APX toxin may be APX1, APX2 or APX3.
The present applicants have found that a microorganism which naturally produces an RTX toxin may be engineered to produce an inactive RTX toxin precursor by eliminating the post-translational activator of the precursor product. Accordingly, in a preferred embodiment the microorganism is unable to produce a post-translational activator of the RTX toxin precursor or produces an inactivated post-translational activator of the RTX toxin precursor. The post-translational activator may be a product of an RTX C gene.
In a preferred embodiment the RTX C gene of the microorganism is inactivated or partially or fully deleted. The RTX C gene may be inactivated by site specific mutagenesis. The RTX C gene may be inactivated by any single or multiple nucleotide substitution, addition and/or deletion. Preferably, the RTX C gene is inactivated by homologous recombination using a targeting construct. The targeting construct may include a selectable marker flanked by sequences homologous to sequences flanking the desired insertion site. The selectable marker may be a gene which confers resistance to a toxic substance such as mercury or may be an antibiotic resistance determinant. The antibiotic resistance determinant may be a gene coding for ampicillin resistance, kanamycin resistance or streptomycin resistance.
In some circumstances it may be undesirable to have a functional antibiotic resistance gene incorporated into a modified microorganism. Accordingly, the present invention contemplates a targeting construct which includes genetic elements, such as repeat sequences, which facilitate excision of the antibiotic resistance gene once the targeting construct has undergone homologous recombination with the host chromosome.
The present invention also contemplates a targeting construct which does not include a selectable marker. For example, the targeting construct may include a segment of the RTX C gene which contains a deletion. Homologous recombination of the targeting construct with the host chromosome may result in the introduction of a deletion into the chromosomal RTX C gene. Selection for recombinants may then be based on the absence of production of the RTX toxin.
The targeting construct may be introduced directly into the host cell in a linear form. Alternatively, the targeting construct may be introduced via a suicide or non-replicating vector. The suicide vector may be any plasmid which does not replicate in the host microorganism. Microorganisms which naturally produce RTX toxins are often non-permissive hosts for pEP vectors. Accordingly, pEP vectors are examples of suicide vectors which may be used in the present invention. The suicide plasmid vector may be pEP-Cxe2x88x92Ampr.
In another embodiment, site specific mutagenesis may be achieved by the technique of plasmid segregation. For example, a plasmid which contains a fragment of an RTX C gene interrupted by a selectable marker gene may be introduced to a microorganism. The microorganism may be subsequently transformed with a second plasmid containing a second selectable marker gene. Host cells containing both plasmids may then be passaged through media which selects only for the second plasmid. Selection for the second plasmid may act against maintenance of the first plasmid. The first plasmid may, therefore, be lost, but in some cases recombination of the interrupted RTX C gene fragment containing the selectable marker into the chromosome may occur. This process therefore may encourage recombination of the interrupted RTX C gene into the chromosomal RTX C gene, thus inactivating the chromosomal RTX C gene.
In a further aspect of the present invention there is provided an expression vector which encodes an RTX toxin wherein said RTX toxin is partially or fully inactivated, said vector encoding an RTX toxin gene including an RTX structural and/or post-translational activator gene wherein said RTX toxin gene is partially or fully inactivated.
The term xe2x80x9cexpression vectorxe2x80x9d as used herein the claims and description includes a chromosomal or extrachromosomal element which is capable of expressing a DNA sequence including a foreign DNA sequence.
The RTX A gene product may be expressed from a chromosomal RTX A gene. The chromosomal RTX A gene may be located in its natural position on the chromosome or may be inserted into the chromosome at a position other than its natural location. In addition, the RTX gene product may be expressed from an RTX A gene located on an extrachromosomal element such as a plasmid. In one embodiment, therefore, an extrachromosomal element containing an RTX A gene may be introduced to a microorganism which has a functional chromosomal RTX A gene and an inactivated chromosomal RTX C gene. The RTX A product expressed from the extrachromosomal element may supplement the RTX A product expressed from the chromosomal gene.
Alternatively, the RTX A gene product may be expressed entirely from an RTX A gene or genes located on extrachromosomal elements such as plasmids. The RTX A genes located on extrachromosomal elements may be expressed either in the presence or absence of selection for the extrachromosomal element. Thus, in one embodiment an extrachromosomal element containing an RTX A gene may be introduced into a microorganism which lacks functional chromosomal RTX A and RTX C genes. The microorganism which lacks functional RTX A and RTX C genes may be produced by mutagenesis of the microorganism. The mutagenesis may result in deletion of the RTX A and RTX C genes or portions thereof.
The extrachromosomal element may be a recombinant expression vector which includes the RTX A gene. Preferably the recombinant expression vector allows expression of the RTX A gene in microorganisms which naturally produce RTX toxins. The recombinant expression vector may allow expression of the RTX A gene in Actinobacillus or related organisms. The recombinant expression vector may be derived from a plG plasmid. The recombinant plasmid may be derived from plG3B. The recombinant plasmid may be plG3B-TIK.
Bacterial vector systems based on APP (Ph) provide an alternative means to deliver xe2x80x9cnaked DNAxe2x80x9d vaccine molecules to host cells. Such naked DNA vaccine/expression systems would include a plasmid capable of replicating in the bacterial system, and a eukaryotic promoter controlling the expression of the foreign/recombinant gene of interest.
In a preferred embodiment the microorganism is able to produce one or more functional proteins which facilitate secretion of RTX toxin molecules. The microroganism may have functional RTX B and/or RTX D genes. In another embodiment, the microorganism is unable to produce at least one of the proteins involved in secretion of RTX toxin molecules or produces at least one inactive protein involved in secretion of RTX toxin molecules. The microorganism may have an inactive RTX B and/or RTX D gene. Thus, the microorganism may be unable to secrete active or inactive RTX toxin molecules.
In a further aspect the present invention provides a recombinant plasmid vector for expression of heterologous proteins in Actinobacillus or related organisms. In a preferred embodiment the recombinant plasmid vector is derived from a naturally occurring plasmid in Actinobacillus pleuropneumoniae. The recombinant plasmid may include a DNA sequence which encodes an RTX toxin precursor. The recombinant plasmid may include an RTX A gene. Alternatively, the recombinant plasmid may include an RTX C gene or a fragment thereof. The RTX C gene or gene fragment may be interrupted by an intervening DNA sequence. The intervening DNA sequence may encode a selectable marker such as an antibiotic resistance gene. The naturally occurring plasmid may be plG3. In a preferred embodiment the recombinant plasmid vector further includes nucleotide sequences which facilitate transfer of DNA to E. coli. The nucleotide sequences may include at least one multiple cloning site and lac gene or portion thereof. The recombinant plasmid vector may be selected from plG317, plG3B or plG3B-T1K.
In another aspect the present invention provides a vaccine composition for inducing an immunological response in a host animal inoculated with said vaccine composition, said vaccine composition including an RTX toxin precursor. The RTX toxin precursor may be an unprocessed expression product of an RTX structural gene. The RTX structural gene may be an RTX A gene.
The present invention further provides a vaccine composition for inducing an immunological response in a host animal inoculated with said vaccine composition, said vaccine composition including a modified microorganism which produces an RTX toxin, wherein said RTX toxin is partially or fully inactivated. Preferably the inactivated RTX toxin is a precursor of an RTX toxin. The precursor may be an unprocessed expression product of an RTX structural gene. The RTX structural gene may be an RTX A gene. Preferably, the microorganism is one which naturally produces an RTX toxin. Preferably the RTX C gene of the microorganism is inactivated or deleted.
In a preferred embodiment the vaccine composition which includes a modified microorganism is a live vaccine.
A vaccine composition of the present invention may be incorporated in any pharmaceutically acceptable vehicle with or without added adjuvants or immunostimulatory molecules.
The adjuvant may be of any suitable type. The adjuvant may be selected from vegetable oils or emulsions thereof, surface active substances, e.g., hexadecylamine, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecyl-ammonium bromide, N, N-dicoctadecyl-Nxe2x80x2-Nxe2x80x2bis (2-hydroxyethyl-propane diamine), methoxyhexadecylglycerol, and pluronic polypols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; immune stimulating complexes (ISCOMS); oil emulsions; and mineral gels and suspensions. A mineral suspension such as alum, i.e. aluminium hydroxide (Al(OH)3), aluminum phosphate or aluminium sulphate is preferred. The adjuvant may be present in amounts of from approximately 1 to 75% by weight, based on the total weight of the vaccine composition.
It will be appreciated that a vaccine according to the present invention, which includes an RTX toxin precursor or a microorganism capable of producing an RTX toxin precursor, has the potential to provide protection against a range of serovars of a microorganism which produces the corresponding RTX toxin.
In another aspect the present invention provides a biological vector including a microorganism which naturally produces an RTX toxin, wherein said microorganism has been modified such that it is incapable of producing an active RTX toxin.
The microorganism which naturally produces an RTX toxin may be selected from Actinobacillus spp, Proteus vulgaris, Morganella morganii, Bordetella pertussis and Escherichia coli. In a preferred embodiment the microorganism is Actinobacillus spp. The Actinobacillus species may be Antinobacillus pleuropneumoniae (APP) and the RTX toxin may be an APX toxin. The APX toxin may be APX1, APX2 or APX3. Preferably, the modified microorganism produces an inactive RTX toxin. Preferably, the inactive RTX toxin is a precursor of the RTX toxin. Preferably, the precursor of the RTX toxin is a product of the RTX A gene.
In a preferred embodiment, the modified microorganism is unable to produce a post-translational activator of the RTX toxin precursor or produces an inactivated post-translational activator of the RTX toxin precursor. The post-translational activator may be a product of an RTX C gene. In a preferred embodiment the RTX C gene of the modified microorganism is inactivated or deleted. The RTX C gene may be inactivated by any single or multiple nucleotide substitution, addition andlor deletion.
The RTX C gene may be inactivated by the introduction of a targeting construct containing a selectable marker into the RTX C chromosomal gene through site specific recombination.The targeting construct may include genetic elements, such as repeat units, which facilitate excision of the antibiotic resistance gene once the targeting construct has undergone homologous recombination with the host chromosome. Alternatively, a targeting construct which does not contain a selectable marker may be used to introduce a deletion in the RTX C chromosomal gene.
The RTX A gene product may be expressed from a chromosomal RTX A gene. The chromosomal RTX A gene may be located in its natural position on the chromosome or may be inserted into the chromosome at a position other than its natural location. In addition, the RTX gene product may be expressed from an RTX A gene located on an extrachromosomal element such as a plasmid. Alternatively, the RTX A gene product may be expressed entirely from an RTX A gene or genes located on extrachromosomal elements such as plasmids.
In a preferred embodiment the microorganism is able to produce one or more functional proteins which facilitate secretion of RTX toxin molecules. The microroganism may have functional RTX B and/or RTX D genes. In another embodiment, the microorganism is unable to produce at least one of the proteins involved in secretion of RTX toxin molecules or produces at least one inactive protein involved in secretion of RTX toxin molecules.
The term xe2x80x9cbiological vectorxe2x80x9d is used in its widest sense to include a biological means suitable for expression of biologically active molecules. The biological means is preferably a viable microorganism although dead organisms could be employed. The biological vector may be non-pathogenic or rendered avirulent or may be given in non-pathogenic or avirulent effective amounts. The term xe2x80x9cbiologically active moleculesxe2x80x9d includes functional molecules such as growth factors, hormones, enzymes, antigens or antigenic parts thereof, cytokines such as interleukins, interferons and tumor necrosis factors. The molecules may be expressed naturally by the biological vector. Alternatively, the molecules may be recombinant molecules expressed by transforming the biological vector with a plasmid carrying a gene or genes encoding the biologically active molecule and which is then expressed; or where the plasmid and/or gene or genes and/or parts thereof are integrated into the host genome, which includes the chromosome and/or any naturally or non-naturally occurring extra-chromosomal element, wherein the gene or genes or parts thereof are expressed.
It will be appreciated that a biological vector of the present invention may be used to provide one or more useful proteins to the host animal. The proteins so provided may act in synergy to bring about an enhanced reaction in the host animal. For example, the biological vector may produce an antigen in combination with a molecule which enhances an immunogenic response in the host animal to the antigen. The molecule which enhances the immunogenic response may be a cytokine.
It will also be appreciated that a biological vector of the present invention may be used to provide a multivalent vaccine. The term xe2x80x9cmultivalent vaccinexe2x80x9d is used in its most general sense and extends to a modified microorganism capable of inducing an immune response to two or more distinct antigenic epitopes on or expressed by the modified microorganism where the two or more epitopes are indigenous to the modified microorganism. More commonly, however, a multivalent vaccine includes a modified microorganism capable of inducing an immune response to virulent forms of said microorganism as well as to heterologous antigens expressed by said microorganism (such as recombinant antigens or those introduced by transduction, conjugation or transformation) and which are not indigenous to the microorganism. In this regard, a multivalent vaccine may be directed to two or more pathogenic agents. Preferred multivalent vaccines are those capable of inducing an immune response against an RTX toxin and to at least one antigenic eptiope from one or more pathogenic agents. The pathogenic agents may be selected from bacterial pathogens such as Haemophilus spp, Serpulina hyodysentedae, Pasteurella spp, Bordetella bronchiseptica, Leptospira spp, Streptococcus spp, Salmonella spp, Escherichia coli, Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae. Alternatively, the pathogenic agents may be selected from viral pathogens such as HCV, PRRSV, PRV, TGEV, PPV.
The present invention further provides a method of producing a modified organism which produces an RTX toxin which is partially or fully inactivated which method includes providing a microorganism which produces an active RTX toxin; and inactivating or deleting the RTX C gene.
The present invention further provides a method of producing a modified organism which produces an RTX toxin which is partially or fully inactivated which method includes providing a microorganism which is incapable of producing an active RTX toxin; and introducing a functional RTX A gene into said microorganism.
The invention in yet a further aspect provides a method for vaccinating an animal against an RTX toxin producing microorganism, said method including administering to said animal an immunologically effective amount of a vaccine in accordance with the present invention.
The method of vaccination may be utilised in the treatment of production animals such as pigs, cattle, sheep, goats. The method of vaccination may also be used in the treatment of companion animals such as horses, dogs and cats. The method of vaccination may also be used in the treatment of humans. In a preferred embodiment the method of vaccination is utilized in the treatment of pigs. Preferably the method of vaccination is utilized in the treatment of porcine pleuropneumonia.
Administration of a vaccine or vaccine vector in accordance with the present invention may be by any suitable route such as by oral or parenteral administration. The administration may be mucosal such as nasal or vaginal. Alternatively, administration may be intramuscular, intradermal, subcutaneous or intraperitoneal. The preparation may be in dry or liquid form. The route of administration chosen may also necessitate additional components such as protease inhibitors, anti-inflammatories and the like.
The invention in yet a further aspect provides a method for vaccinating an animal against a pathogenic organism, said method including administering to said animal an effective amount of a vaccine vector in accordance with the present invention wherein said vaccine vector synthesises an immunologically effective amount of an antigen of said pathogenic organism.
In yet another aspect the present invention provides a method for the production of an inactive RTX toxin which method includes culturing a modified microorganism in accordance with the present invention and recovering the inactive toxin produced by said microorganism. The inactive RTX toxin produced by this method may, for example, be used as the active immunogen in a vaccine for stimulating a protective immune response against an RTX toxin.
Throughout the description and claims of this specification, the word xe2x80x9ccomprisexe2x80x9d and variations of the word, such as xe2x80x9ccomprisingxe2x80x9d and xe2x80x9ccomprisesxe2x80x9d, is not intended to exclude other additives, components, integers or steps.
In order that the invention may be more readily understood we provide the following non-limiting examples.